The anti-inflammatory cytokine interleukin-37 (IL-37) belongs to the IL-1 family but is not expressed in mice. We used a human IL-37 (hIL-37tg) expressing mouse, which has been subjected to various models of local and systemic inflammation as well as immunological challenges. Previous studies reveal an immunomodulatory role of IL-37, which can be characterized as an important suppressor of innate immunity. Here, we examined the functions of IL-37 in the central nervous system and explored the effects of IL-37 on neuronal architecture and function, microglial phenotype, cytokine production and behavior after inflammatory challenge by intraperitoneal LPS-injection. In wild-type mice, decreased spine density, activated microglial phenotype and impaired long-term potentiation (LTP) were observed after LPS injection, whereas hIL-37tg mice showed no impairment. In addition, we crossed the hIL-37tg mouse with an animal model of Alzheimer’s disease (APP/PS1) to investigate the anti-inflammatory properties of IL-37 under chronic neuroinflammatory conditions. Our results show that expression of IL-37 is able to limit inflammation in the brain after acute inflammatory events and prevent loss of cognitive abilities in a mouse model of AD. Neuroinflammation is characterized by glial cell activation and is mediated via pro-inflammatory signals . In geneInnate immune responses and neuroinflammation are thought to contribute significantly to the progression of AD . TherefoFor example, the release of IL-1β under pathological conditions can lead to deficits in learning and memory processes as well as in long-term potentiation (LTP) . In addiUnlike most members of the IL-1 family that induce inflammation, this important cytokine family also includes two members (IL-37 and IL-38) that act as anti-inflammatory signalling agents and are thought to dampen an ongoing innate immune response . In partAn important factor for the expression of IL-37 related to the instability sequence was also discovered in a mouse cell line. In the present study, we used hIL-37tg mice to investigate the effect of IL-37 expression on the acute inflammatory processes induced by LPS or IL-1β administration. In addition, we crossed the APP/PS1 mouse model of AD with hIL-37tg mice to investigate the role of IL-37 expression during the chronic state of neuroinflammation and the consequences for the progression of AD pathology.To investigate the effect of IL-37 on microglial cell activation, the release of the pro-inflammatory cytokines IL-6, IL-1β, and TNF-α was analyzed after LPS stimulation of wild-type (WT) and IL-37 transgenic microglial cells .wt/tg) and homozygous (IL-37tg/tg) IL-37 transgenic primary microglia was analyzed and compared to IL-6 secretion of WT microglia upon LPS stimulation. The results showed that the amounts of IL-6 secreted by microglial cells from homozygous IL-37 transgenic (IL-37tg/tg) mice were significantly reduced compared to microglial cells from control mice after 24 hr of stimulation with 100 ng/ml and 1 µg/ml LPS and additionally also 6 hr after stimulation with 1 µg/ml LPS =7.941 p=0.007; p=0.007; p=0.054; F=3.915 p=0.052; p=0.043; p=0.359; F=12.74 p=0.002; p=0.004; p=0.009) (wt/tg) also showed decreased IL-6 secretion, but only after 6 hr of stimulation with 1 µg/ml LPS =12.74 p=0.002; p=0.009) (tg/tg) IL-37 transgenic mice (referred to as IL-37tg). Further analysis also confirmed the reduction in inflammatory mediators by measuring the release of IL-6 when other concentrations of the inflammatory stimulus were examined =2.629 p=0.05509; p=0.0171, p=0.0086, p=0.0238)  . Microglp=0.009) . Thus, texamined  and coulexamined  and IL-1examined , compare=0.0238) . As ment=0.0238) . Examini=0.0238) , confirmTo investigate the potential effect of recombinant IL-37 (rIL-37) in inhibiting LPS-induced release of proinflammatory cytokines (here IL-6) by microglia, primary WT microglial cells were pretreated with either 100 ng/mL or 500 ng/mL rIL-37 for 2 hr and then stimulated with increasing concentrations of LPS. Pretreatment with rIL-37 was chosen because microglial cells from IL-37tg mice had very low basal expression of IL-37 even in the absence of LPS . TherefoTaken together, these results indicate that microglial cells in particular release lower levels of pro-inflammatory cytokines upon acute inflammatory stimulation in the presence of IL-37.Recent studies have discovered several metabolic intermediates that contribute directly to immune function. Among them, the TCA cycle-related metabolites succinate and itaconate are novel markers associated with pro-inflammatory macrophage activation and function . To addr+/CD45low population) =19.52 p=0.0045; p=0.0017; p>0.9999; F=48.42 p=0.0004; p=0.0001) (+/CD45high population) showed no changes in CD68-expressing cells after LPS stimulation or between genotypes (-/CD45high population)  homozygous IL-37tg mice (referred to as IL-37tg) and age-matched control animals with either saline (vehicle control group) or LPS (2x0.5 mg/kg) . Both ex=0.0001)  compared=0.0001) . Remarka=0.0001) . Further=0.0001) . Further=0.0001) , whereasenotypes . In addienotypes  and, in enotypes  and leukulation) , again hThe activation feature of microglial cells is reflected in the total number of microglial cells in the brain parenchyma and the number of primary processes of these CNS resident immune cells. For example, a higher number of IBA-1-positive cells and a reduced number of primary processes correlate with increased microglial activation . TherefoNeuroinflammation has been shown to impair hippocampal network function . BecauseTo assess the effects of IL-37 on neuronal structure, we analyzed the dendritic spine density of hippocampal neurons from all experimental groups. Dendritic spines are small protrusions representing the postsynaptic part of excitatory synapses and were counted on the apical dendrites of CA1 neurons as well as on the dendrites of dentate gyrus neurons. A significant decrease in the density of dendritic spines of CA1 pyramidal neurons and dentate gyrus cells was observed in control mice treated with LPS compared with saline-treated mice =22.81 p<0.0001; p<0.0001; p=0.1145; F=26.68 p<0.0001; p<0.0001; spines DG F=3.307 p=0.0805; p=0.0062; p>0.9999; F=0.529 p=0.4718; p=0.0295) .Similar to the LTP data, the density of dendritic spines of neurons from IL-37tg animals was unchanged when LPS- and saline-injected mice were evaluated . In concGiven the possibility that peripheral immune cells in IL-37tg mice also produce IL-37 after LPS challenge and already attenuate the systemic immune response, the question arose whether IL-37 would have a similar protective effect directly in the CNS after LPS stimulation. To accurately demonstrate the beneficial effect of IL-37 in the CNS without the influence of peripheral cells, another electrophysiological experiment was performed.Here, acute hippocampal slices from control and IL-37tg animals were prepared and stimulated with LPS (10 µg/ml) in ACSF for 2 hr after a resting period before recording. In the control groups, the sections were kept in ACSF only. These further results indicated that direct IL-37 expression in the acute slices of IL-37tg mice appears to be sufficient to reverse the impairments in LTP after LPS administration. This is because, in contrast to the significant impairment of LTP after LPS administration in acute hippocampal slices of control mice =4.925 p 0.05) , no impaTo investigate the anti-inflammatory properties of the recombinant IL-37 (rIL-37) protein in vivo, wild-type mice were pretreated with either 300 ng rIL-37 per animal (i.p.) or an equivalent amount of vehicle  for 3 consecutive days. On day 4, animals were injected with 60 ng i.p. IL-1β or saline as control . After aIn the absence of rIL-37 pretreatment, we detected a significant performance deterioration in IL-1β-injected mice compared to the corresponding saline-injected control group =18.25 p=0.0052; p=0.0046; p=0.7313) . In contTo investigate the possible preventive effect of rIL-37 on LPS-induced neuroinflammation, in addition to IL-1β-immunostimulation, WT mice were pretreated with either 100 ng rIL-37 per animal (i.p.) or an equivalent amount of vehicle  for 3 consecutive days. On day 3 and 4, the animals were injected twice with LPS (0.5 mg/kg) and 3 hr after the last injection, the level of proinflammatory cytokines in the brain was measured . The resOur results demonstrated the protective properties of IL-37 on microglial activation, production of pro-inflammatory mediators, impairment of cognition, and disruption of long-term potentiation (LTP) after acute immunostimulatory challenge. To investigate the potential of IL-37 to attenuate chronic inflammation, we next analyzed transgenic APP/PS1 mice, which serve as a widely used animal model for Alzheimer’s disease , and cro+/CD45low cell population in the brain) was analyzed at different ages of mice =14.39 p=0.0008; p=0.0008; p=0.0529; p=0.0494) =13.45 p=0.0009; p=0.0007; p=0.0263; p=0.0985; FACS 20–23 m F=13.38 p=0.0009; p=0.0007; p=0.0359; p=0.0383)  . The mic=0.0383) . However=0.0383) . We then=0.0383)  of APP/P=0.0383)  and that=0.0383)  compared=0.0383) .To further analyze whether the observed lower Aβ plaques in APP/PS1-IL37tg were due to higher Aβ uptake, an Aβ uptake assay was performed to examine phagocytic activity in APP/PS1 and APP/PS1-IL37tg mice carrying the CX3CR1-GFP gene (heterozygous) . For thiThe APP/PS1 mouse model of Alzheimer’s disease shows cognitive deficits as early as 8 months of age . To inveThe observed improvement of learning and memory in APP/PS1-IL37 transgenic mice prompted us to analyze whether hippocampal network function could also be improved by this genotype. Therefore, we measured synaptic plasticity at the Schaffer collateral pathway as described above in these animals. Acute hippocampal slices from APP/PS1 mice showed significant deficits in LTP compared to corresponding slices from age-matched control mice =9.286 p=0.0004) . This waTo determine the rescued phenotypes observed in APP/PS1-IL37tg mice at the cellular level, neuronal morphology of the CA1 subregion of the hippocampus was analyzed in all experimental groups. A significant reduction in the density of dendritic spines was observed in 9–12 month-old APP/PS1 mice compared to age-matched control mice =6.318 p=0.0032; p=0.0026) . HoweverSaa2, which is highly expressed in response to inflammation and tissue injury , indicating increased expression of the potential receptor for IL-37 in microglia after systemic LPS challenge  in microglial cells from WT and IL-37tg mice. The 500 most significantly expressed genes were shown in a heat map . This hie injury , Vgf, whytokines , Bcl6b, response , Il23a,  disease , and Icae system . These gsyndrome . The Kcnophrenia . The Kifactivity . In addi or IL-1 , Mmp3, wtivation , Acod1, ammation , and thehallenge .Although this mRNA expression assay for microglia showed that there were no significant differences between the overall gene profile of microglia from WT and IL-37 after LPS challenge, a specifically lower microglial response was nevertheless detectable in IL-37tg mice, which may shed light on the immunomodulatory role of IL-37 in acute and chronic neuroinflammation.The regulation of inflammation is an extremely complex and tightly controlled signaling event in which cytokines crucially modulate the process . The intThe acute inflammatory response was analyzed in a mouse model of septic shock in which increased levels of IL-6, TNF-α, and IL-1β produced by activated microglia were detected . The abiRemarkably, the Warburg effect describes the change in metabolism toward the production of ATP from anaerobic glycolysis to oxidative phosphorylation observed in macrophages by increased mTOR levels and decreased AMPK activity after LPS stimulation . It was To confirm the anti-inflammatory effect of IL-37 in acute inflammatory conditions in vivo, further studies were performed by systemic LPS injection in control and IL-37tg animals. Inflammation and activation of microglia are crucial hallmarks of various neurological diseases . On the The resulting neuroinflammation and activation of microglial cells after peripheral LPS challenge also lead to functional and structural changes in the neuronal network, particularly affecting long-term potentiation (LTP)  and spinOur results suggest decreased neuroinflammation induced by systemic immune stimulation in the presence of IL-37. However, the anti-inflammatory effect of IL-37 in the CNS may be a secondary effect due to the reduction of the inflammatory response in the periphery. To identify the direct possibility of IL-37 expression in the CNS without peripheral influence, further electrophysiological experiments were performed with acute hippocampal slices from control and IL-37tg mice. Administration of LPS to the acute slices of IL-37tg mice suggests that IL-37 expression directly in the CNS may also be responsible for the reversal of neuronal impairments after peripheral LPS challenge. In addition, we confirmed the mechanisms of anti-inflammatory action in IL-37tg mice by using the recombinant IL-37 in WT mice. Recombinant human IL-37 has protective effects against endotoxemia, acute lung and spinal cord injury, asthma, and myocardial infarction . Next, wIn this study, we specifically analyzed the activation status of microglial cells as a neuroinflammatory feature leading to structural changes in the neuronal network. Although our data suggest that IL-37 most likely transmits its signals via microglia, astrocytes also play an important role in maintaining neuronal circuits . ActivatGpr84  and the in vivo model here, because primary microglia showed upregulation of itaconate upon LPS treatment. Finally, RNA sequencing data showed upregulated gene expression of the IL-18 receptor, suggesting that expression of the IL-18 receptor on microglial cells may be responsible for IL-37 signal transduction by microglia in IL-37tg mice. It is worth noting that the lack of large differences in microglial gene expression profiles between WT and IL-37tg mice may be due to the timing of microglial isolation, because we isolated microglia 3 hr after the last LPS injection, which was also the case for the other experiments. However, the timing of gene regulation is different from that of protein synthesis. Therefore, it can be assumed that 3 hr after the last LPS injection, when we detected the deleterious effects, the transcripts of the responsible genes have already been degraded.To better predict the role of microglia in IL-37 signal transduction after immunostimulation, single-cell type RNA sequencing was performed. Analysis of mRNA gene expression profiles revealed that approximately 11014 genes were differentially expressed in microglia after systemic LPS administration, clearly confirming the close communication between the peripheral immune system and the CNS. Although no significant differences were found between the microglial gene profile of WT and IL-37tg mice, however, these data indicated the specific gene profile in the both genotypes after the LPS challenge. For example, some genes were up- or down-regulated in the WT mice, which was the case to a lesser extent in the IL-37tg mice. Among them were some important genes such as Saa2 , Vgf Bu, Bcl6b , Il23a , Icam1 , and Tk2Gpr84 , Mmp3 . Moreover, these results suggest that rIL-37 may play an important future role as a therapeutic intervention in many neurological diseases associated with inflammatory responses. However, further experiments with recombinant IL-37 need to be performed to obtain a detailed picture of the mechanism underlying rIL-37 signaling. A recent study has shown that the efficacy of IL-37 is limited when administered systemically, suggesting a beneficial effect on local CNS cells rather than cells in the periphery . FurtherTo analyze chronic neuroinflammation, we used APP/PS1 mice as a model for Alzheimer’s disease (AD). AD is the most common form of dementia, and in 2015, an estimated 46.8 million people worldwide were living with the dementia . ResearcIt is noteworthy that previous studies have shown that IL-1β and IL-6 play an important role in the progression of AD, as both were released from microglial cells surrounding Aβ plaques in AD patients and animal models . FurtherIn general, the putative mechanism of IL-37 signaling in neurodegenerative diseases is not clear. Translation to humans might suggest IL-37 as a biomarker for inflammatory diseases with changes in IL-37 levels in patients associated with these diseases . IL-37 aIn summary, during chronic neuroinflammation associated with Alzheimer’s disease, a significant deficit in synaptic plasticity  was documented, whereas rescue was observed in transgenic AD animals overexpressing the anti-inflammatory cytokine IL-37. In addition, a significant effect on the formation of amyloid-β plaques, a pathological feature of AD, was documented, suggesting that attenuation of neuroinflammation combined with increased clearance is sufficient to produce beneficial effects on learning and memory. Indeed, the results reported here demonstrate that expression of an anti-inflammatory IL-37 cytokine is able to reduce neuroinflammation and cognitive decline in a mouse model of Alzheimer’s disease. Furthermore, recent studies in spinal cord injury  and in aAll experiments were performed and analyzed blinded to the experimenter.All animals used in this study were of either sex, equal distributed to the experiments with exception in the behavioral test were only males were used . Mice weEscherichia coli  was used. The systemic immune stimulation with LPS was performed by intraperitoneal injection. The body weight of the stimulated animals was monitored to determine the appropriate volume of LPS. Animals were either injected with 2x0.5 mg/kg LPS or 0.9% sodium chloride (NaCl) as a control.In all performed experiments related to any lipopolysaccharide (LPS) stimulus, the same LPS from To investigate the acute effect of LPS-induced direct immunostimulation and subsequent local IL-37 expression in the CNS, LPS administration was performed during electrophysiological recordings. For this purpose, acute hippocampal slices from adult control and IL-37tg mice were pretreated with LPS (10 µg/ml) 2 hr before and throughout the recording period.Wild-type mice were injected intraperitoneally with 300 ng of recombinant IL-37 per mouse for 3 consecutive days, followed by an injection of 60 ng of recombinant IL-1β per mouse.To test the preventive effect of rIL37 with LPS, WT mice received an intraperitoneal injection of 100 ng of rIL-37 or vehicle for three consecutive days. Two hours after the third injection, mice received an injection of either 0.5 mg/kg LPS or 0.9% sodium chloride (NaCl) as a control, followed by a second injection 24 hr later.2 at 37 °C for 2–3 weeks. After 3 days incubation, the media was replaced 50% with fresh media. In the following every 7 days the media was replaced completely. After 2–3 weeks, the culture has reached confluence and the flasks were shaken at 180 rpm for 3 hr at 37 °C. The media including the microglia cells was collected without disrupting the astrocyte layer on the bottom of the flask and was centrifuged at 3000 rpm for 10 min at room temperature (RT). Microglia cells were plated in 96-well plate with a density of 6x104 cells/well and were treated with different concentrations of LPS for 24 hr. In the last hour of the treatment, ATP (5 mM) was added to the cells.Neonatal mouse brains (P3 – P5) were used for culture preparation as shown previously . Briefly5 cells/well) and maintained for 24 hr followed by a 100 ng/ml LPS stimulus.To prepare a primary astrocyte culture, neonatal mouse brains (P3-4) were used. After removal of the brain, the hippocampus and meninges were carefully removed. The cortices were transferred on ice to fresh HBSS 1 x, and the tissue was homogenized using a 10 ml pipette. The HBSS was then washed off, and the brains were placed in a dissociation solution containing DNAse for 30 min at 37 °C, and the remaining tissue pieces were further dissociated by pipetting. After centrifugation at 800 rpm for 7 min, the supernatant was removed and the cells were resuspended in culture medium  and placed on a 40 µm cell strainer and finally transferred to a T-75 flask coated with poly-D-lysine. When cells reached confluence, they were shaken overnight at 220 rpm to remove other glial cells, and then astrocytes were passaged. The cells were passaged three times until the experiments were performed. Experiments were performed on day in vitro (DIV) 15–19. Astrocytes were plated in a 12-well plate  using the GentleMACS (Miltenyi Biotec) program Protein_01. After centrifugation at 4000 g for 5 min at 4 °C the supernatant was again centrifuged for 10 min at 13,000 g at 4 °C. Brain homogenates (1:2) (diluted in 1% BSA solution) were analyzed using R&D systems ELISA Kits.Enzyme-linked immunosorbent assay (ELISA) was used to quantify cytokines in either brain homogenates or supernatants of treated primary microglia cells. Mice were deeply anesthetized with COTo quantify the expression of IL-37 in primary microglia, cells were treated with 100 ng/mL LPS for 24 hr for seven different time periods . Total RNA was isolated using peqGOLD TriFast. cDNA was prepared using BioLabs ProtoScript II First Strand cDNA Synthesis Kit.For tissue extraction the RNA purification Kit (Macherey-Nagel) was used. cDNA was prepared by using the High Capacity cDNA Reverse Transcription Kit (Thermofisher).IL-37: 5´-GGG AGT TTT GTC TCT ACT GTG AC-3´(forward) and 5´-CCC ACC TGA GCC CTA TAA AAG-3´(reverse); GAPDH 5´-GCC TTC CGT GTT CCT ACC-3´(forward) and 5´-CCT CAG TGT AGC CCA AGA TG-3´(reverse). Expression levels of target mRNA was analyzed using the ∆∆Ct method and were normalized to the expression level of the house keeping gene GAPDH, which was used as an internal control.Then, real-time quantitative PCR (qPCR) was performed using BlueProbe qPCR Mix (Biozym) and the following primer pairs: To check the amount and morphology of microglial cells in the brain tissue, brains were isolated and fixed in 4% paraformaldehyde (PFA) for 24 hr and then cryoprotected in 30% sucrose solution in phosphate buffered saline (PBS 1 x) for 24 hr. Samples were stored in Tissue-Tek O.C.T. compound (A. Hartenstein Laborversand) at –70 °C. 30 µm brains sections were cut using the Cryostat. Using the free floating method these slices were washed in 1 x PBS and blocked in 1 x PBS solution containing 0.2% Triton X-100, 10% goat serum and 1% BSA for 1 hr at room temperature (RT). Slices were incubated overnight at 4 °C with anti-ionized calcium-binding adaptor molecule 1 (IBA-1)  primary antibody diluted in 1 X PBS, 0.2% Triton X-100 and 10% goat serum. Cy3-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L)  was used as secondary antibody diluted in 1 X PBS. Sections were washed as before and stained with 4’,6-diamidino-2-phenylindole (DAPI) (SIGMA) followed by cover-slipping with Fluoro-gel with Tris buffer (Electron Microscopy Sciences).Microglia activation was analyzed by measuring the marker CD68 with the FACS method . Single The phagocytic activity regarding Aβ uptake in microglial cells of APP/PS1 and APP/PS1-IL37tg animals was analyzed by FACS. Here, WT, APP/PS1 and APP/PS1-IL37tg animals was used and crossed with the Cx3Cr1-GFP mouse line. The Cx3cr1-GFP transgenic mice express a microglia-specific (or monocytic-specific) green fluorescent protein in the CNS. To visualize Aβ particles animals were injected intraperitoneal with 10 mg/kg methoxy-XO4  in 50% DMSO/ 50% NaCl (0.9%) 3 hr prior to the start of experiments. After the brain was removed, the procedure was as in section ‘FACS analysis’.There are several methods to evaluate cognitive function in rodents. Of these, one method is called ‘Y-Maze’ in which a mouse is placed in a maze with three equal arms (hence the term ‘Y’) each 32 cm from the center. First the mouse is free to explore the first two arms while the third arm is blocked. After the mouse get to know the existence of the two arms during 3 min training, the mouse was returned to its cage for 1.5 hr. Then the mouse is placed back to the Y-Maze with all three arms available. The observer records how often the mouse enters an arm. Due to animal’s natural tendency to explore a newly introduced environment it is expected to be expressed by a high frequency of spontaneous alteration performance (SAP). The SAP score is a triplet of three successive different arm visits .The Morris water maze (MWM) test is an assay to analyze spatial memory formation and retention . A circuThe mice were trained in the Morris water maze for 8 days with the invisible platform located in the northwest (NW) quadrant. Each day, animals were placed from randomly starting positions  for 4 trials in the water with 5-min intervals. The animals had a maximum of 60 s to find the platform otherwise they were guided to the platform and allowed to stay for additional 15 s. The memory retention was measured by performing a reference test. One reference test was performed on day 3 of the training acquisition (prior to the training session) and another reference test 24 hr after the last training day (day 9). The platform was removed during this reference test. The animals were tracked for 45 s (starting position SE).2, killed by decapitation following fast brain removal and transfer into ice-cold carbogenated (95% O2 and 5% CO2) artificial CSG (ACSF) containing 124 mM NaCl, 4.9 mM KCl, 1.2 mM KH2PO4, 2.0 mM MgSO4, 2.0 mM CaCl2, 24.6 mM NaHCO3 and 10 D-glucose, pH 7.4. Acute hippocampal slices were prepared (400 µm) using a tissue chopper. The hippocampal slices were placed into an interface chamber (Scientific System Design) and incubated at 32 °C in a constant flow rate (0.5 ml/min) of carbogenated ACSF for 2 hr. Afterwards the recordings were started and field excitatory post synaptic potentials (fEPSPs) were measured in the stratum radiatum of the hippocampus CA1 sub-region. The Schaffer collateral pathway was stimulated using two monopolar, lacquer-coated stainless-steel electrodes . Long-term potentiation (LTP) was induced after 20 min baseline recording by theta-burst stiulation (TBS) including four bursts at 100 Hz repeated 10 times in a 200ms interval. This stimulation was repeated three times in a 10 s interval. Only healthy sections with a stable baseline were included in the data set. Using the IntraCell software  the data set was analyzed.To study learning and memory processes on cellular level, we did electrophysiological recording experiments in CA1 hippocampal neurons as described before . Briefly2 and sacrificed by decapitation. The brain was incubated in FD rapid Golgi-Cox staining kit according to the manufacturer’s protocol. The tissue was incubated in a mixture of solution A (potassium dichromate and mercuric chloride) and B (potassium chromate) for at least 14 days at RT in the dark. After incubation of A-B the tissue was placed for 1 week in solution C (tissue protection) at RT. In the following, the brain was blocked in 2% agar and 200-µm-thick coronal sections were cut using a vibratome (Leica VT 1000 S). The slices were collected on gelatin-coated glass slides and stained with solution D and E before being dehydrated through graded alcohols and mounted using Permount (Thermo Fisher Scientific).To analyze the morphology of hippocampal neurons, Golgi-Cox staining was performed like previously described . Briefly2 and sacrificed via decapitation. Brains were isolated and hemispheres were fixed in 4% PFA for 24 hr and then cryoprotected in 30% sucrose solution in phosphate buffered saline (PBS 1 x). Hemispheres were stored in Tissue-Tek (Hartenstein Laborversand) at –70 °C and cutted using the Leica Cryostat (CM3050 S) in 30 µm slices. The sections were transported on gelatin-coated slides and in the following stained with the Congo-Red manufactorer’s protocol using the FD Congo-Red Solution kit  and mounted with Permount (Thermo Fisher Scientific).To examine the Aβ-plaques in APP/PS1 and APP/PS1-IL37 mice plaques were analyzed for the amount and size using the Congo-Red staining. Mice were deeply anesthetized with COTo analyze the hippocampal neuron morphology, CA1 and dentate gyrus (DG) cells were imaged in the three-dimensions (z-stack thickness of 0.5 µm) using Axioplan 2 imaging microscope (Zeiss) with a 63 x (N.A. 1) oil objective equipped with a digital camera . All selected dendrites were analyzed per spine density via number of spines  per micrometer of dendritic length more than 50–60 µm which were positioned at least 40–50 µm away from the cell soma.To analyze microglia morphology microscopic images of anti-IBA-1 were taken within the area of cortex and hippocampus. Images were taken in 3D (z-stack thickness 1 µm) using Axioplan 2 imaging microscope (Zeiss) equipped with an ApoTome module (Zeiss) with a 20 X objective  and digital camera . IBA-1 positive cells were counted with clearly visible nuclei by DAPI staining for microglia density and the processes of IBA-1 positive cells were analyzed to investigate the activation status of these cells by using the ImageJ software .2.To survey the Aβ-plaques images from Congo-Red stained slices were taken. Congo-Red staining presents a bright fluorescence emission at 614 nm with excitation at 497 nm. Images of brain sections were taken using an Axioplan 2 imaging microscope (Zeiss) with a 2.5 X objective (N.A. 0.07) connected to a digital camera (Nikon) with the same light exposure time of 1 s in all groups. Plaque load and plaque size were analyzed using the ImageJ software  with the analyze particle tool. The polygon selection tool was used to generate the region of interest (ROI) for the area of hippocampus and cortex. Plaque load (number of particles) and plaque size (area of particles) were normalized to the area of hippocampus and cortex and plotted as plaque load and plaque size per mm5 cells) were plated onto 12-well plates and incubated for 24 hr. The medium was exchanged with fresh medium or medium mixed with 10 ng/ml LPS for 24 hr. To extract intracellular metabolites cells were washed once with 0.9% NaCl and 500 µL of a cold methanol/water mixture was added. The water fraction contained the internal standard (IS) D6-glutaric-acid (c=1 µg/ml). Cells were scraped and transferred into 250 µL of –20 °C Chloroform. After vortexing samples for 20 min at 4 °C with maximal speed in an automatic shaker, samples were centrifuge for 5 min at above 15,000 xg in a table centrifuge (4 °C). A total of 200 µL of the upper phase was transferred in a GC glass vial with micro insert and evaporated to dry under vacuum at 4 °C, overnight. To avoid condensation, the GC glass vials were warmed to room temperature under vacuum and capped afterwards with magnetic caps.Primary microglia cells  with settings according to log file . Differential gene expression was calculated by R package ‘edgeR’. Functional analysis was performed by R package ‘clusterProfiler’ . The cDNA synthesis was performed and libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina at the Genome Analytics of the Helmholtz Center for Infection Research. The libraries were sequenced on Illumina NovaSeq 6000 using NovaSeq 6000 S1 Reagent Kit  with an average of 5x107 reads per RNA sample. Before alignment to reference genome (mm10) each sequence in the raw FASTQ files were trimmed on base call quality and sequencing adapter contamination using Trim Galore! wrapper tool. Reads shorter than 20 bp were removed from FASTQ file . Trimmedlog file . Featurelog file . Only gerofiler’ .Sample size for all experiments were calculated a priory with G*Power software (HHU Düsseldorf). Samples were allocated into the experimental groups randomly.Data were analyzed and plotted by GraphPad Prism 8  and presented as mean ± SEM. Statistically analysis were performed with either unpaired t-test, one-way ANOVA (post hoc test Fisher’s LSD or Turkey’s multiple comparisons) or two-way ANOVA (post hoc test Fisher’s LSD or Bonferroni’s multiple comparisons) depending on experiments. The minimum significance value was considered as p<0.05 . In this manuscript, the authors demonstrated that acute and chronic neuroinflammation was attenuated in human IL-37 (hIL-37) transgenic mice, thus revealing the effects of an anti-inflammatory cytokine hIL-37 in the central nervous system of mice. This study will be of interest to scientists studying neuroinflammation and searching for potential therapeutic targets. public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Jeannie Chin as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "IL-37 expression reduces acute and chronic neuroinflammation and rescues cognitive impairment in an Alzheimer's disease mouse model" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:1. The expression profile of hIL-37 receptors on different cell types under different conditions should be shown. The phenotype seen in microglia in vitro was attributed to hIL-37 acting on microglia themselves, but it is not clear this is the only possibility.2. Experiments using human cell lines or patient samples would provide additional validation.3. The authors mentioned that in acute inflammation, little LPS is likely to enter the brain because of an intact blood-brain barrier (BBB). It is also possible that the peripherally-induced inflammation may contribute to the neuroinflammation centrally. Due to the systemic overexpression of hIL-37, to exclude the peripheral effects of hIL-37, the experiments of direct injection of hIL-37 into the brain would help clarify the conclusion.4. Background, Lines 87,88: The overall premise that anti-inflammatory therapies will be beneficial in AD is overly simplistic. The inflammatory activation of glial cells is required as a protective immune response, including pro-inflammatory activation. Also, retrospective studies that have demonstrated that NSAIDs are not a successful therapeutic strategy go against this notion. Please consider rephrasing.5. What is known about IL-37 expression in humans? Since it is expressed by all cells in the current model, will this hamper biological interpretation, since the brain RNAseq database suggests that it is highly enriched specifically in microglia? This confounding factor makes biological interpretation of the in-vivo data a little difficult.6. Results, Page 4, Lines 148-152: Description of Figure 1C-1E is confusing and unclear as written. Please consider rephrasing.7. Results, Page 5: The points about the TCA cycle metabolites is raised in this section for the first time. Please consider introducing this concept in relation to AD / related immune responses in the introduction.8. Figure 3C: Please include what the X-axis is plotting? As a supplemental figure, please include your initial gating strategy used for all flow cytometry data. Are there any CD68+CD11b+ CD45 high cells in these animals? If an effect of IL-37 is seen in filtration of peripheral inflammatory macrophages into the brain, this is a very strong data set to improve the impact of this figure.9. Results, page 7: The authors make the statement that IL-37 signaling has a significant impact on the pro-inflammatory activation of microglia in the brain. However, this is based on just the measurement of IL-1b and microglial numbers. The authors should measure other markers of inflammation that are known to go up in the brain upon LPS challenge. Maybe using an Elisa based multiplex assay such as MSD or Luminex to measure brain inflammatory cytokines will add to this data set.10. Results, page 7: Figure 4A-4B: Was the LPS injection used for these experiments the same as outlined for figure 3? Please clarify this in the method section, especially since 2 different LPS treatment paradigms have been used for 4A-4B and 4G-4H.11. The paradigm for the Y-maze testing , though in-line with the current study, is not biologically relevant. Since IL-37 will be upregulated as part of the neuroinflammatory response, have the authors tried to treat animals with IL-37 after IL-1b or concurrently with IL-1B?12. Figure 1B: This figure suggests that 1 i.p injection with rIL-1B completely abolishes any working memory In WT mice. This is surprising. Please clarify.13. For Figures6 and 7: Were both sexes used for APPPS1 studies? If so, please clarify in the methods section as to the number of males and females, given that female APPPS1 mice show more aggressive disease as compared to males.14. Figure 6. Please show all gating strategies as well as relevant gates drawn to identify CD11b+ CD45low microglia, as well as the methoxy X-04+ vs methoxy X-04- microglia. Is there a CD11b+ CD45high microglial subset at this age, since at earlier timepoints, these distinct microglial subsets are evident and have different activation profiles.15. Please include details of how brain tissue was processed to prepare single-cell suspension for all FACS data. i.e: percoll based density gradients or myelin removal beads etc?16. Authors discuss their methoxy-X04 data as indicative of "increased clearance" of plaques. Careful interpretation of this data set is required as this assay only measures Abeta "uptake". Since no measures of clearance have been assessed, please reword and/ reinterpret this in the results and Discussion section.17. Please include representative images for plaque loads in the cortex that correlate with quantification in 6I. Also, please include a representative image for differences in plaque size.18. Figure 7A: Please mention the average age of the animals used in the 3 animal groups for MW testing. 9-12 months is a big age range for these animals and results will be skewed if one cohort is largely composed of older animals vs. younger.19. The Discussion section is rather long and largely used to re-summarize results. The authors should reconsider using this section to put their data into context for the big-picture field of neurodegeneration. Some suggestions – How do the authors picture IL-37 signaling to play a role in human AD?a. Given the detailed description of distinct microglial subsets associated with amyloid vs. tau pathology in AD, do the authors anticipate IL-37 to have distinct effects depending on pathological outcomes?b. Literature has demonstrated that IL-1b, IL-6 and TNFa from reactive microglia may play a role in exacerbating tau-spread. The authors should discuss the implications of their findings in this lightc. Is anything is known about IL-37 signaling in other neuroinflammatory models ? A possible discussion of these in the context of neuroprotection may strengthen this section.20. in vitro phenotypes in Figure 1 were not closely connected with the following in vivo neuroinflammation phenotypes. Although three different neuroinflammation models were used, the detailed mechanisms for how IL-37 reduced neuroinflammation were not well addressed.21) Figure 3 and Figure 5 showed reduced pro-inflammatory cytokine production in brain lysates. However, glial cells other than microglia, such as astrocytes and infiltrating leukocytes could be sources of inflammatory cytokines. There was no evidence supporting that reduced cytokine levels were intrinsic to microglia as modelled in the in vitro system. Thus, in vitro and in vivo phenotypes appeared somewhat disconnected.22) In Figure 5, IL-1β-induced neuroinflammation model was used to demonstrate the beneficial effects of IL-37 on cognition and synaptic function. Could injection of recombinant IL-37 display similar effects for LPS challenge model in Figure 3 and Figure 4?23) Reduced IL-37-mediated neuroinflammation was shown in both chronic and acute models in the manuscript. Did microglia or other cells play a major role in those models? The detailed mechanisms regarding cell type contribution were largely missing in the manuscript.24. Figure 2 showed differential metabolomic profiling of microglial cells between WT and IL-37tg mice. However, there was no further evidence demonstrating that the metabolic function of microglial cells was indeed altered by IL-37 expression. It would be better to show the results of seahorse or other metabolic functional assays.25. The models for acute neuroinflammation including LPS and IL-1β challenge were systematic inflammation. It might be reasonable to propose that reduced neuroinflammation was a secondary effect to reduced inflammation response in the periphery. In addition, in Figure 3, would injection of LPS twice induce tolerance responses?26. In Figure 3 and Figure 6, CD68 was used as an activation marker for microglia. However, CD68 expression by itself is not enough to define microglia to be in the activation state. The phenotypic changes of microglial cells would also depend on specific models used. Additional experimental evidence is needed for defining the reduced activation status of microglia in IL-37tg mice. Essential revisions:1. The expression profile of hIL-37 receptors on different cell types under different conditions should be shown. The phenotype seen in microglia in vitro was attributed to hIL-37 acting on microglia themselves, but it is not clear this is the only possibility.It has been shown that IL-37 requires the receptors IL-18Rα and IL-1R8 (SIGIRR) to exert its versatile anti-inflammatory effects in innate signal transduction. Based on the previous studies now listed in the revised manuscript, astrocytes are also capable of expressing the required receptors for IL-37. However, in the new experiments we performed, we stimulated primary astrocytes with LPS and then measured the levels of IL-6 and TNF-α . In contrast to primary microglia from IL-37tg mice , levels of pro-inflammatory cytokines were not reduced in primary astrocytes from IL-37tg mice after 100ng/ml LPS stimulation for 24 hours . Therefore, we can assume that although astrocytes also express receptors for IL-37, IL-37 signalling may not occur via them because the anti-inflammatory consequences are not expressed. These new experiments are added in the supplementary section and discussed in the revised manuscript.A new supplementary Figure 1 is added. The changes can be found in lines 118-124 and 171-175, respectively.2. Experiments using human cell lines or patient samples would provide additional validation.Although the suggestion to use human material could significantly strengthen the results of this manuscript, performing new experiments with human cell lines requires a large time window because our laboratory currently has no experience with this. In addition, obtaining patient samples requires official ethical approval, which takes a lot of time. Therefore, in the revised manuscript, in the Introduction and Discussion, we try to refer to the recent human studies on the role of IL-37 in various human diseases such as autism and multiple sclerosis.The changes can be found in lines 120-124 and 731-734, respectively.3. The authors mentioned that in acute inflammation, little LPS is likely to enter the brain because of an intact blood-brain barrier (BBB). It is also possible that the peripherally-induced inflammation may contribute to the neuroinflammation centrally. Due to the systemic overexpression of hIL-37, to exclude the peripheral effects of hIL-37, the experiments of direct injection of hIL-37 into the brain would help clarify the conclusion.In this study, we used IL-37tg mice that received i.p. injection of LPS to investigate the potential anti-inflammatory effects of IL-37 on the acute deleterious inflammatory response induced by LPS in the brain. We agree with the comment that containment of the inflammatory response in the periphery by IL-37 could prevent the inflammatory consequences in the brain, and we were not sure whether local expression of IL-37 in the brain could be beneficial in blocking the effects of LPS. Therefore, in a separate ex vivo approach, we treated the acute hippocampal slices of IL-37tg mice with LPS and then measured long-term potentiation (LTP) using electrophysiological experiments. Although LTP was impaired when acute slices from WT mice were treated with LPS, this was not the case for slices from IL-37tg mice. Subsequent mRNA expression in acute slices showed that IL-37 expression was higher in slices from IL-37tg mice after LPS stimulation . Thus, we can conclude that in addition to peripheral IL-37, local induction in the brain during LPS administration may be part of the rescue mechanisms. In our revised manuscript, we have made this point more explicit.The information can be found in lines 291-308.4. Background, Lines 87,88: The overall premise that anti-inflammatory therapies will be beneficial in AD is overly simplistic. The inflammatory activation of glial cells is required as a protective immune response, including pro-inflammatory activation. Also, retrospective studies that have demonstrated that NSAIDs are not a successful therapeutic strategy go against this notion. Please consider rephrasing.As the editor mentioned, although acute inflammation is part of the body's response to tissue damage and is critical to the healing process, a prolonged inflammatory response can be harmful. Chronic inflammation has been shown to affect and accelerate each of the three hallmarks of AD pathology, including Aβ accumulation, tau phosphorylation, and cognitive decline associated with loss of synapses and neurons. Therefore, attenuating chronic inflammatory processes may be a useful way to treat or prevent the progression of AD. NSAIDs are generally used for their anti-inflammatory, analgesic, and antipyretic effects. NSAIDs work by blocking the production of prostaglandins by inhibiting two cyclooxygenase enzymes. However, prostaglandins contribute to the development of the major signs of acute inflammation. Therefore, as the editor mentioned, they are not a useful therapy to prevent the progression of AD as a result of a chronic inflammatory response.We have taken this point into consideration and reworded the mentioned part in the introduction to make it clearer.The information can be found in lines 89-91.5. What is known about IL-37 expression in humans? Since it is expressed by all cells in the current model, will this hamper biological interpretation, since the brain RNAseq database suggests that it is highly enriched specifically in microglia? This confounding factor makes biological interpretation of the in-vivo data a little difficult.Previously, IL-37 was shown to be constitutively expressed in various human tissues and cells, which may help in maintaining immune homeostasis. IL-37 is expressed in human immune cells mainly in circulating monocytes, tissue macrophages, dendritic cells, tonsil B cells, and plasma cells. In addition, IL-1β and TNF-α were shown to increase the expression of IL-37 in cultured human microglia. Review of the expression profile of human iPSC derived microglia after LPS treatment using the GEO database also reveals the presence of IL-37(https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE186301), but it is still not clear which cell types in the human brain produce IL-37 abundantly. In the revised manuscript, the previously known information about IL-37 in humans was added in the Introduction and Discussion sections.The information can be found in lines 123-127 and 550-554.6. Results, Page 4, Lines 148-152: Description of Figure 1C-1E is confusing and unclear as written. Please consider rephrasing.We followed the editor’s advice and rephrased this part.7. Results, Page 5: The points about the TCA cycle metabolites is raised in this section for the first time. Please consider introducing this concept in relation to AD / related immune responses in the introduction.The relevant information was added in the revised manuscripts. The information can be found in lines 131-136.8. Figure 3C: Please include what the X-axis is plotting? As a supplemental figure, please include your initial gating strategy used for all flow cytometry data. Are there any CD68+CD11b+ CD45 high cells in these animals? If an effect of IL-37 is seen in filtration of peripheral inflammatory macrophages into the brain, this is a very strong data set to improve the impact of this figure.All detailed information on plot axis and gating strategies was added to the revised manuscript. In addition, the FACS data were reanalyzed to verify the populations of infiltrating macrophages expressing CD68 in the brains of WT and IL-37tg mice after LPS challenge. Supplementary Figure 3 is included in the manuscript. The corresponding information can be found in lines 227-238.9. Results, page 7: The authors make the statement that IL-37 signaling has a significant impact on the pro-inflammatory activation of microglia in the brain. However, this is based on just the measurement of IL-1b and microglial numbers. The authors should measure other markers of inflammation that are known to go up in the brain upon LPS challenge. Maybe using an Elisa based multiplex assay such as MSD or Luminex to measure brain inflammatory cytokines will add to this data set.In this study, we specifically analyzed IL-1ꞵ because the production of this cytokine depends on the activation of the NLRP3 inflammasome, which also includes IL-18 and the closely related IL-37; all of these cytokines belong to the IL-1 family. This cytokine is an important mediator of the inflammatory response, particularly upon LPS stimulation . Therefore, we focused on this selected known cytokine.However, in the new experiment, we also measured the levels of other cytokines, including IL-6 and TNF-α, using Elisa and added the data to the revised manuscript in supplementary Figure 2.For microglia, we also analyzed CD68 expression, which in combination with proinflammatory cytokines (IL-1ꞵ) in brain and cultures represent features of high activation and neuroinflammatory processes.These points have been highlighted in the revised manuscript. The information can be found in lines 227-230.10. Results, page 7: Figure 4A-4B: Was the LPS injection used for these experiments the same as outlined for figure 3? Please clarify this in the method section, especially since 2 different LPS treatment paradigms have been used for 4A-4B and 4G-4H.Because Figure 4A-C are the electrophysiological experiments after LPS challenge in vivo, the timing of the experiment is the same as in Figure 3, but we have made this clearer in the revised manuscript. The methods and information for Figure 4G-I have been included in the Methods and Results sections. The information can be found in lines 291308 and 909-914.11. The paradigm for the Y-maze testing , though in-line with the current study, is not biologically relevant. Since IL-37 will be upregulated as part of the neuroinflammatory response, have the authors tried to treat animals with IL-37 after IL-1b or concurrently with IL-1B?Although we agree with the comment that the best therapeutic regimen for the anti-inflammatory agent is to use it after the onset of symptoms, in this part of the study we focused on recommending rIL-37 as a preventive strategy rather than as a therapeutic agent for acute inflammation. In addition, pro-inflammatory members of the IL-1 family were hypothesized to play an important role in the pathophysiology of sepsis, as serum levels of these cytokines were greatly increased in patients with sepsis. Moreover, injection of IL-1β elicits an acute and rapid response in the body (similar to septic shock), making the search for preventive strategies for these conditions of great importance compared with therapeutic strategies, because the immune response is very rapid and not gradual. We have tried to make this point clearer in the revised manuscript.12. Figure 1B: This figure suggests that 1 i.p injection with rIL-1B completely abolishes any working memory In WT mice. This is surprising. Please clarify.Acute systemic inflammation has been shown to cause deficits in working memory early in the disease course in dementia models. Moreover, systemic inflammation primarily impairs attention and cognitive flexibility, including working memory, rather than associative learning. Therefore, short-term memory formation may be more vulnerable to even mild inflammatory responses, as it has been shown that a low-dose injection of LPS, which elicits a very mild immune response and small changes (< 1 °C) in core body temperature, severely impairs working memory . This is not the case for long-term memory formation. We therefore hypothesized that a systemic injection of IL-1β, which induces strong systemic inflammation, can completely abolish working memory, which is also confirmed by our results. These points have been carefully highlighted in the Discussion section. The information can be found in lines 590-597.13. For Figures6 and 7: Were both sexes used for APPPS1 studies? If so, please clarify in the methods section as to the number of males and females, given that female APPPS1 mice show more aggressive disease as compared to males.Mainly male mice were used in this study. All detailed information about the sexes of the mice was clarified in the revised manuscript. The information can be found in lines 885886.14. Figure 6. Please show all gating strategies as well as relevant gates drawn to identify CD11b+ CD45low microglia, as well as the methoxy X-04+ vs methoxy X-04- microglia. Is there a CD11b+ CD45high microglial subset at this age, since at earlier timepoints, these distinct microglial subsets are evident and have different activation profiles.All detailed information on gating strategies is included in the revised manuscript (lines 382-393). Cx3cr1GFP mice were used in this particular experiment. Therefore, the gating strategies were performed for the positive FITC cell population. The detailed information on this and supplementary Figure 6 are included in the revised manuscript.15. Please include details of how brain tissue was processed to prepare single-cell suspension for all FACS data. i.e: percoll based density gradients or myelin removal beads etc?All detailed information on the preparation of single cell suspensions for FACS data has been accurately added to the revised manuscript. The information can be found in lines 994-997.16. Authors discuss their methoxy-X04 data as indicative of "increased clearance" of plaques. Careful interpretation of this data set is required as this assay only measures Abeta "uptake". Since no measures of clearance have been assessed, please reword and/ reinterpret this in the results and Discussion section.We fully agree with this comment. The explanation and interpretation of this experiment was adjusted accordingly in the revised manuscript. The information can be found in lines 382-393 and 682-684.17. Please include representative images for plaque loads in the cortex that correlate with quantification in 6I. Also, please include a representative image for differences in plaque size.All the representative images mentioned for specific brain regions and different plaque sizes were added to the revised manuscript. The information can be found in Supplementary Figure 5.18. Figure 7A: Please mention the average age of the animals used in the 3 animal groups for MW testing. 9-12 months is a big age range for these animals and results will be skewed if one cohort is largely composed of older animals vs. younger.Carefully age-matched mice were used in all experimental groups. Detailed information on the average age of the animals was included in the revised manuscript. The information can be found in Supplementary Figure 8.19. The Discussion section is rather long and largely used to re-summarize results. The authors should reconsider using this section to put their data into context for the big-picture field of neurodegeneration. Some suggestions – How do the authors picture IL-37 signaling to play a role in human AD?a. Given the detailed description of distinct microglial subsets associated with amyloid vs. tau pathology in AD, do the authors anticipate IL-37 to have distinct effects depending on pathological outcomes?b. Literature has demonstrated that IL-1b, IL-6 and TNFa from reactive microglia may play a role in exacerbating tau-spread. The authors should discuss the implications of their findings in this lightc. Is anything is known about IL-37 signaling in other neuroinflammatory models ? A possible discussion of these in the context of neuroprotection may strengthen this section.All of the above valuable points were included in the Discussion section of the revised manuscript.20. in vitro phenotypes in Figure 1 were not closely connected with the following in vivo neuroinflammation phenotypes. Although three different neuroinflammation models were used, the detailed mechanisms for how IL-37 reduced neuroinflammation were not well addressed.Although this study suggests the positive role of IL-37 in modulating microglial activations as a possible key mechanism in controlling neuroinflammation, the detailed mechanisms are not addressed in this manuscript. However, we believe that our findings obtained in various inflammatory models clearly demonstrate the beneficial role of IL-37 and thus may pave the way for future studies to discover the detailed mechanisms and possible neuroprotective potential of IL-37, especially with regard to therapy in humans. The detailed possible underlying mechanisms of the neuroinflammatory potential of IL37 were proposed in the Discussion section of the revised manuscript based on the recent literature examining the beneficial role of IL-37 in autism, MS and spinal cord injury.In addition, we performed a single-cell RNA sequencing experiment in the microglial cell population of both WT and IL-37tg mice after LPS challenge. We selected several differentially expressed candidate genes that may be important as key molecules to decipher the IL-37 underlying mechanisms in the future. Figure 8 and supplementary Figure 7 are included in the revised manuscript.21) Figure 3 and Figure 5 showed reduced pro-inflammatory cytokine production in brain lysates. However, glial cells other than microglia, such as astrocytes and infiltrating leukocytes could be sources of inflammatory cytokines. There was no evidence supporting that reduced cytokine levels were intrinsic to microglia as modelled in the in vitro system. Thus, in vitro and in vivo phenotypes appeared somewhat disconnected.As noted by the reviewer, the role of other cells in the brain, including astrocytes, brain infiltrating macrophages, and lymphocytes, is not evident in the cytokine assay of brain lysate. However, reanalysis of our FACS data with gating strategies for infiltrating macrophages and lymphocytes revealed no differences between groups, suggesting a smaller population and no significant activation profile compared with microglia. Moreover, based on the new experiment performed with primary astrocyte cultures (see answer to question 1), the key role of microglia in this scenario is suggested. The relevant information was added to the manuscript (lines 171-175) and can be found in supplementary Figure 1 and 3.22) In Figure 5, IL-1β-induced neuroinflammation model was used to demonstrate the beneficial effects of IL-37 on cognition and synaptic function. Could injection of recombinant IL-37 display similar effects for LPS challenge model in Figure 3 and Figure 4?As mentioned by the editor, confirmation of the potential anti-inflammatory role of IL-37 in our in vivo model with LPS challenge is of great importance. We performed new experiments and pre-treated the mice with rIL-37 followed by LPS challenge. Subsequently, the level of pro-inflammatory cytokines in the brain was measured using Elisa. The new data were added to the revised manuscript. The information can be found in the manuscript (lines 335-346) and in supplementary Figure 4.23) Reduced IL-37-mediated neuroinflammation was shown in both chronic and acute models in the manuscript. Did microglia or other cells play a major role in those models? The detailed mechanisms regarding cell type contribution were largely missing in the manuscript.The detailed information on the importance of microglial function in neuroinflammatory processes and in the progression of Alzheimer's disease, based on the most recent findings, was added to the revised manuscript.In addition, the new experiments on the role of astrocytes and other infiltrating immune cells in this scenario were added to the revised manuscript. Please see the answers to questions 1 and 21.24. Figure 2 showed differential metabolomic profiling of microglial cells between WT and IL-37tg mice. However, there was no further evidence demonstrating that the metabolic function of microglial cells was indeed altered by IL-37 expression. It would be better to show the results of seahorse or other metabolic functional assays.Acod1 gene specifically in microglia, which is responsible for itaconate production, was also added to the revised manuscript. There was no significant differences in Acod1 expression after LPS challenge between WT and IL-37tg mice, which may be due to the degradation of this transcript that has already occurred . The information can be found in the manuscript (lines 622-636) and in supplementary Figure 7.In this study, we focused on the two metabolites that have been shown to be associated with inflammation in macrophages. We fully agree with the reviewers that further experiments showing metabolic alterations could be useful to address the subsequent changes in more detail. However, it has already been pointed out that itaconate and succinate in particular are involved in inflammation, especially in macrophages. Further data on the expression level of the 25. The models for acute neuroinflammation including LPS and IL-1β challenge were systematic inflammation. It might be reasonable to propose that reduced neuroinflammation was a secondary effect to reduced inflammation response in the periphery. In addition, in Figure 3, would injection of LPS twice induce tolerance responses?We agree with the reviewer that containment of the inflammatory response in the periphery by IL-37 could prevent the inflammatory consequences in the brain (please see the answer to question 3).As mentioned in the comment, the immune tolerance phenomenon is very important in this study. The protocol for 2-times LPS injection was adopted from the study by Wendeln et al. 2018. Their results clearly showed that tolerance in the peripheral immune response occurred after the 2-times LPS injection, as indicated by the cytokine expression levels in the serum. Remarkably, however, this was not the case for the CNS, as cytokine levels in the brain were significantly increased after the second LPS injection.26. In Figure 3 and Figure 6, CD68 was used as an activation marker for microglia. However, CD68 expression by itself is not enough to define microglia to be in the activation state. The phenotypic changes of microglial cells would also depend on specific models used. Additional experimental evidence is needed for defining the reduced activation status of microglia in IL-37tg mice.CD68 is a lysosomal protein that is highly expressed by activated macrophages and activated microglia and expressed to a very low extent by resting microglia, as evidenced by much of the literature . Therefore, CD68 can be considered a well-established activation marker for microglia. However, in this study, our results additionally showed a lower number of IBA-1-positive cells and lower IL-1β expression in the brain of IL-37tg mice after LPS challenge, which may confirm the reduced activation status of microglia in IL-37tg mice. The relevant literature on CD68 as an activation marker for microglia were added to the revised manuscript.


BCOR (BCL6 corepressor)-rearranged sarcoma (BRS) is a rare sarcoma entity with a predominantly BCOR-CCNB3 fusion. In this paper, we present an index case of BRS with a novel BCOR-CLGN  gene fusion that was first identified by next-generation sequencing and then verified by Sanger sequencing. We also carried out in silico protein analysis to demonstrate the 3D structure of the chimera protein. We concluded that, due to its heterogeneity, molecular ancillary tests serve as powerful tools to discover such unusual variants. The fusion protein used in the in silico analysis is an appropriate approach to understanding the exact pathogenesis of such a rare variant.BCOR (BCL6 corepressor)-rearranged sarcomas (BRSs) are a heterogeneous group of sarcomas previously classified as part of the group of “atypical Ewing” or “Ewing-like” sarcomas, without the prototypical ESWR1 gene translocation. Due to their similar morphology and histopathological features, diagnosis is challenging. The most common genetic aberrations are BCOR-CCNB3 fusion and BCOR internal tandem duplication (ITD). Recently, various new fusion partners of BCOR have been documented, such as MAML3, ZC3H7B, RGAG1, and KMT2D, further increasing the complexity of such tumor entities, although the molecular pathogenetic mechanism remains to be elucidated. Here, we present an index case of intrathoracic BRS that carried a novel BCOR-CLGN  gene fusion, exhibited by a 52-year-old female diagnosed initially by immunohistochemistry due to the positivity of a BCOR stain; the fusion was identified by next-generation sequencing and was confirmed by Sanger sequencing. In silico protein analysis was performed to demonstrate the 3D structure of the chimera protein. The physicochemical properties of the fusion protein sequence were calculated using the ProtParam web-server tool. Our finding further broadens the fusion partner gene spectrum of BRS. Due to the heterogeneity, molecular ancillary tests serve as powerful tools to discover these unusual variants, and an in silico analysis of the fusion protein offers an appropriate approach toward understanding the exact pathogenesis of such a rare variant. BCOR (BCL6 corepressor) expression has been found in a variety of tumor types, including clear cell sarcoma of the kidney, endometrial sarcoma, rhabdomyosarcoma, and central nervous system and myeloid tumors [BCOR-rearranged small blue round cell sarcoma (BRS), which commonly occurs in the bones of young patients, represents a rare soft tissue tumor entity and shares morphological similarities with Ewing sarcoma (ES) without the EWSR1 rearrangement described first by Pierron et al. [BCOR gene is located in the Xp11.4 chromosome region; it facilitates BCL6-regulated transcriptional repression via epigenetic signal transduction mechanisms [BCOR bounding to the polycomb-group RING finger homolog (PCGF) of polycomb-repressive complex 1 (PRC1) [HOX group genes [BCOR participates in one of the non-canonical PRC1 complexes, PRC1.1 [Aberrant d tumors ,2,3. Amon et al. . The BCOchanisms ,6 to medchanisms . The epi1 (PRC1) , via the1 (PRC1) . PRC1 fuup genes ,10. BCORIn a previous study, Wang et al. found that the presence of BCOR at BCOR-responsive targets is crucial for the recruitment of the PRC1.1 complex, the maintenance of H3K27me3, and the recruitment of cPRC1 complexes. Without the C-terminal domain, the protein does not interact with KDM2B, PCGF1, and RNF2; the C-terminus of BCOR is both necessary and sufficient for BCOR targeting. The linker and PUFD regions are critical for recruiting BCOR and maintaining polycomb domains at the targets .BCOR aberrations in BRS are internal tandem duplication (ITD) and translocation, causing gene fusion; for the latter, the most common fusion partner is CCNB3 in the Xp11.22 chromosome region due to paracentric inversion, which links the BCOR coding sequence to exon 5 of the CCNB3 gene. The additional stretch of amino acids (ITD) in the PUFD domain might interfere with PCGF1 binding and, thus, could affect PRC1-related epigenetic modifications [The most common ications ,14,15.MAML3, ZC3H7B, RGAG1, and KMT2D [BRS is currently classified under the category of “undifferentiated small round cell sarcoma of soft tissue and bone”, based on the latest WHO classification (2020) and represents approximately 15% of cases within this category. Despite the morphological resemblance to Ewing sarcoma (ES), the genomic and transcriptomic profiles of BRSs showed that they are genetically distinct entities . Neverthnd KMT2D ,18,19.BCOR FISH, RNA isolation, RT, Sanger sequencing, an NGS gene panel analysis targeting 54 genes , and in silico protein analyses were performed. Here, we present a novel case of intrathoracic soft tissue somatic carried BCOR-CLGN  gene fusion, which further expands the genetic diversity of BRS. The CLGN gene encodes the calmegin protein, but its oncological significance is currently unknown. The aims of our study were (i) to present the histological and immunohistochemical (IHC) staining features in a case of BRS with unusual intrathoracic manifestation, (ii) to design a custom RNA-based NGS panel, including genes that may be involved in the pathogenesis of these rare entities of sarcomas, (iii) to identify somatic gene mutations (SNVs and indels) and gene fusions using NGS, (iv) to confirm the detected translocation using fluorescence in situ hybridization (FISH), (v) to verify gene fusions after reverse transcription (RT) using classic bidirectional Sanger sequencing, and (vi) to perform an in silico protein analysis to predict its function in the detected fusion protein. For this purpose, histologic examination, IHC, EWSR1 rearrangement being detected by FISH were investigated. Due to its rarity, we found only one case that met these criteria. The retrospective patient population is derived from the Department of Pathology at the University of Debrecen, from the period of January 2015–December 2022. Cases diagnosed as “atypical Ewing sarcoma” without For research purposes, this patient consented and agreed, according to the ethical guidelines of the University of Debrecen, to the use of tissue blocks and clinical information for this report. All protocols have been approved by the authors’ respective Institutional Review Boards for human subjects (IRB reference number: 60355-2/2016/EKU and IV/8465-3/2021/EKU). This study was managed according to the Declaration of Helsinki.Hematoxylin and eosin (H&E)-stained slides were analyzed by pathology specialists. Histological evaluation and interpretation were conducted according to WHO classification.After the sectioning of 4 μm slides from formalin-fixed, paraffin-embedded (FFPE) blocks, deparaffinization in xylene, and rehydration in a series of decreasing concentrations of ethanol were performed. Antigen retrieval using either the Bond Epitope Retrieval Solution 1 (pH~6) or the Bond Epitope Retrieval Solution 2 (pH~9)  was carried out at 99–100 °C for 20–30 min. The slides were then treated with cytokeratin , WT-1 , NUT , CD56 , BCOR , CD99 , INSM1 , NKX2.2 , SATB2 , TLE1 , SS18-SSX , Brg1 , desmin , and MIB-1  separately. Immunostaining was conducted with the Leica BOND-MAX™ autostainer and the peroxidase/DAB Bond™ Polymer Refine detection system  was utilized for visualization purposes.EWSR1  and BCOR  on the X chromosome, from the BACPAC Resources Center, Emeryville, CA, USA) break-apart probes separately, using a modified manufacturer’s protocol. Deparaffinized sections  were treated with protease solution . Slide and probe co-denaturation was carried out at 75 °C for 10 min, while hybridization was at 37 °C in a moist chamber for 18 h . Post-hybridization washes were performed with 2× saline–sodium citrates (SSC) for 5 min. The slides were then washed with 0.4× SSC at 74 °C for 3 min and 2× SSC/0.05% Tween 20 for 2 min. After washing, the nuclei were counterstained with 4′-6′ diamidino-2-phenylindole . Scoring was performed using a Zeiss Axio Imager Z2  fluorescence microscope. The images were captured and analyzed using ISIS software v.5.5.4. . FISH was performed on 5 µm-thick sections of the FFPE block, with Genomic tumor-derived RNA (tdRNA) was extracted from FFPE tissues using the ReliaPrep FFPE Total RNA Miniprep System  according to the manufacturer’s instructions. The concentration of tdRNA was measured with the Qubit™ RNA HS Assay Kit, using a Qubit 4.0 fluorimeter .EWSR1 and BCOR genes, was applied to identify the SNVs, indels, and gene fusions. This solution uses anchored primers with known translocation partners and reverse primers that hybridize into sequencing adapters to detect breakpoints and partners. A total of 100–250 ng of RNA was loaded into the assay. cDNA was taken after the first-strand synthesis and run in a quantitative RT-PCR pre-sequencing QC assay. The pre-sequencing quantitative reverse-transcription PCR assay was performed in duplicate on cDNA, using primers targeting the control gene transcripts of VCP and using 35 cycles to determine the amount of intact RNA in a given sample. The final libraries were quantified with a KAPA library quantification kit , diluted to a final concentration of 4 nM, and pooled by equal molarity.For NGS library preparation, an RNA-based Archer FusionPlex custom gene panel  specific for sarcomas, including the For sequencing on the MiSeq System , the libraries were denatured using 0.2 nM NaOH and diluted to 40 pM with hybridization buffer . The final loading concentration was 8 pM libraries and 5% PhiX. Captured libraries were sequenced in a multiplexed fashion with a paired-end run, to obtain 2 × 150 bp reads, with a depth of coverage of at least 500×. Sequencing was conducted according to the MiSeq instruction manual. Trimmed fastq files were generated using the MiSeq reporter  and were uploaded to the Archer Analysis v7 website . For alignment, the human reference genome GRCh37  was built. Translocations were stated at a fusion sequence of over 5 reads, with reads from gene-specific primers comprising at least 10% of the total reads. Gene fusion frequency was calculated for both the fusion transcript reads and the total reads ratio.BCOR fusion, detected by NGS, and knowing the breakpoints, Sanger sequencing was performed. To exclude the possibility of an analytical mistake, another RNA isolation method was also used to fulfill the Sanger sequencing . RNA quality was determined via eukaryote total RNA nano assays.For confirming the ® III First-Strand Synthesis Kit . RT-PCR was performed using the Advantage-2 PCR kit  for 32 cycles at an annealing temperature of 64 °C. Two pairs of primers were used for excluding analytical problems, which were as follows: BCOR exon 15 forward primers: 5′-GGTGGAATTCACGAACGAAA-3′ (F1), 5′-GAATTCACGAACGAAATTCAGA-3′ (F2), and CLGN exon 9 reverse primers: 5′-GGATAAATTTTGGTTCATCATCAAG-3′ (R1) and 5′-ATCATCAAGCCAGCCAGCA-3′ (R2). The lengths of the first and second PCR products were 170 and 150 bp, respectively. The amplified products were purified and sequenced using the Sanger method.For Sanger sequencing, a reverse transcription-polymerase chain reaction (RT-PCR) was carried out. The cDNA quality was tested for the phosphoglycerate kinase 1 (PGK1) housekeeping gene (247 bp of amplified product). One microgram of total RNA was used for cDNA synthesis with a SuperScript Protein information for the BCOR  and CLGN  proteins were obtained from the UniProt database and the RCSB Protein Data Bank. The translated BLAST:blastx web program was used to blast our nucleotide sequence ,27,28,29The instability index gives an estimate of a protein’s stability in vitro. The aliphatic index of a protein is regarded as a positive factor for the growth of thermostability of globular proteins and is specifically defined as the relative volume occupied by aliphatic side chains . The GRAVY score is calculated as the sum of the hydropathy values of all the amino acids, divided by the number of residues in the sequence. The 3D structure of the chimera protein was built using the Robetta RoseTTAFold   protein t-test was performed to compare the IUPred3 scores between the chimera and wild-type BCOR PUFD domain. A one-way repeated measures (RM) ANOVA was performed to compare the relative ASA (0–1) and ASA (Å2) between chimera, AF predicted and experimentally determined the wild-type BCOR PUFD domains. Tukey’s multiple comparison tests were applied to determine the differences between the experimentally determined (PDB ID: 4HPL) and modeled (AlphaFold) structures of the wild-type PUFD domain and the PUFD domain of the chimera protein (Robetta).A paired-sample The index case was a 52-year-old female with a previous medical history of hysterectomy, conducted in 2021 due to an endometrial tumor . One year after the operation, at the postoperative follow-up, a right-side chest wall tumor was found, and positron emission tomography-computed tomography (PET-CT) revealed a right parasternal 5 × 4.5 × 3.5 cm-sized high metabolic-rate tumor with costal cartilage, pectoral muscle, and pleural invasion A. The reMicroscopically speaking, the H&E-stained slides showed hyper-/hypocellular small blue round cell proliferation in a lobulated pattern within the fibromyxoid-vascular stroma A. InfiltEWSR1 FISH examination was carried out and no rearrangement of the EWSR1 gene was identified, excluding the possibility of Ewing sarcoma. Based on the BCOR positivity via an immunohistochemical stain, the BCOR break-apart FISH assay was performed with a positive result, indicating BCOR gene rearrangement . The BCOR breakpoint was located in exon 15, while the CLGN breakpoint was affected in exon 9. Nucleotide alterations were not proven. To confirm the BCOR-CLGN fusion and identify the breakpoints, RT was followed by Sanger sequencing. The bidirectional electropherogram of the fusion genes is presented in To analyze not only the SNVs and indels but also gene fusions, NGS panels that are specific for 54 genes identified in sarcomas were applied. BCOR-CLGN fusion gene; two proteins were found in BLASTX with a 100% match: NP_001116857.1:1–1755 BCOR  and NP_001124147.1:295–610 calmegin precursor . Translated BLAST:blastx was performed on the cDNA sequence of the BCOR gene (TG) and one nucleotide of the CLGN gene (G) encode Trp with the TGG codon; this Trp in the 1755th position corresponds to the wild-type C-terminal residue of the BCOR protein. At the breakpoint, the 1766th residue of the chimera protein is the 296th Asp of calmegin.The full-length wild-type BCOR (UniProt ID: Q6W2J9) and calmegin (UniProt ID: O14967) proteins consist of 1755 and 610 residues, respectively. The BCOR-calmegin chimera protein consists of a total of 2070 residues, encompassing 1–1755 residues of BCOR and 296–610 residues of calmegin . Upon fuThe physicochemical properties of the fusion protein sequence were calculated using the ProtParam web server tool. It was found that the fusion protein is composed of 2070 residues, the calculated molecular weight is 228 kDa, and the pI is 5.05. The total number of negatively charged residues (D + E) was 295, and the total number of positively charged residues (R + K) was 203 .The molecular weight of the chimeric protein was 15.8% and 69.3% higher than those of the wild-type BCOR and calmegin proteins, respectively. The total hydrophobicity of the chimera protein was similar, with a 3.97% and 5.38% change compared to the wild-type proteins. Nevertheless, the aliphatic index and instability index values of the chimera and the wild-type BCOR proteins are similar , while the difference is notably higher in the case of calmegin; the aliphatic index was 7.10% lower, while the instability index was 18.97% higher for the chimera. Overall, the predicted stabilities of the wild-type BCOR and the chimera proteins were highly comparable .The region of interest for the analyses was 1634–1748 because this is where the BCOR’s PUFD domain is located. We performed secondary structure prediction using the GORIV web server, but it did not show any considerable changes; the predicted secondary structure of the PUFD domain was identical in the wild-type BCOR and the chimera. The 296–610 region of calmegin was also predicted to have the same arrangement of secondary structural elements, even in the chimera protein.We used IUPred3 to predict the disorder propensities of the regions of the PUFD domain in the wild-type and chimera proteins A. We pret-test was performed to compare the IUPred3 scores (0–1) (p < 0.0001.A paired-sample es (0–1) B betweenTo predict the putative 3D structure of the chimera protein, we used the Rosetta program to build a 1400 residue-long region of the complete chimera protein (671–2070 amino acids). p < 0.0001). Tukey’s multiple comparison tests revealed that the mean score was significantly different between the PUFD-BCOR-AlphaFold vs. PUFD-chimera-Robetta  and PUFD-BCOR-4HPL.pdb vs. PUFD-chimera-Robetta . There was no statistically significant difference in mean scores between the PUFD-BCOR-AlphaFold and the PUFD-BCOR-4HPL.pdb (0.7953).The comparison of the ASA values of PUFD domains in wild-type (PUFD-BCOR-AlphaFold and PUFD-BCOR-4HPL.pdb) and chimera proteins (PUFD-chimera-Robetta) was carried out . An RM o2) between at least two groups  = 14.18), p < 0.0001). Tukey’s multiple comparison test found that the mean score was significantly different between the PUFD-BCOR-AlphaFold vs. the PUFD-chimera-Robetta , and PUFD-BCOR-4HPL.pdb vs. chimera PUFD domain . There was no statistically significant difference in mean scores between the PUFD-BCOR-AlphaFold and the PUFD-BCOR-4HPL.pdb (p = 0.8547)  C.2 and relative ASA 0.3368 means were reduced in the PUFD-chimera-Robetta compared to PUFD-BCOR-AlphaFold 70.63 Å2, 0.4061 and PUFD-BCOR-4HPL.pdb, 69.23 Å2, 0.3972. It was also found that there was no significant difference in either ASA Area (Å2) or relative ASA, between the PUFD-BCOR-4HPL.pdb and the PUFD-BCOR-AlphaFold  in exon 15 and CLGN (chr4:141.317.359) in exon 9, respectively. To validate this finding, we performed RT-PCR and Sanger sequencing, which also revealed the in-frame fusion of both BCOR and CLGN and further verified our novel finding.BRS was originally reported to exhibit nversion . Later, B, MAML3 , KMT2D [3 [KMT2D , and, reD [RGAG1 . The oncBCOR gene has 16 alternative exons that regulate germinal center formation in lymph nodes and apoptosis. BCOR is also known to be a member protein of polycomb repressive complex 1.1 (PRC1.1) through its PUFD domain, in exon 15, which interacts with the RAWUL domain of the PCGF protein of PRC1.1 [BCOR were largely preserved, it might contribute a significant oncogenic role by exerting its genetic silencing effect via epigenetic regulation [CLGN encodes a protein called calmegin, which is a testis-specific endoplasmic reticulum chaperone protein that plays an important role in spermatogenesis, intracellular calcium homeostasis, and the synthesis of proteins and steroid hormones. It is usually expressed in the testis, prostate, and heart. It has been documented that calmegin is transcriptionally regulated by histone deacetylase and CpG methyltransferase [CLGN translocation/fusion-associated tumor, based on our best knowledge, has not been reported as yet. We assume that the BCOR-CLGN chimeric protein reported herein may exert its oncogenic potential via the antiapoptotic effect of BCOR and epigenetic dysregulation from the calmegin.It is known that the f PRC1.1 , while if PRC1.1 ,44. In ogulation . On the nsferase ; the dernsferase ,49,50,51BCOR-CLGN fusion gene is composed of full-length BCOR protein (1–1755) and the 295–610 region of calmegin; the chimera protein consists of a total of 2070 residues. Using the ProtParam tool, the physicochemical characteristics of the wild-type BCOR protein and fusion protein were calculated and compared. By a disorder prediction using the IUPred3 web server, we found a significant increase in the PUFD domain’s disorder propensity in the chimera protein, compared to the wild-type BCOR protein’s domain. Using the Robetta webserver, a 1400 AA long region of the 2070 AA long chimera protein (encompassing its 671–2070 residues) was built up; this proposed structure was used to perform alignments to compare the folding of the chimera to those of the wild-type BCOR and calmegin proteins. The crystal structure of the PUFD domain (PDB ID: 4HPL) was also compared to the PUFD-chimera-Robetta and PUFD-BCOR-AlphaFold structures to visualize the structural differences and similarities of the domain (We found that the protein product of the e domain . One lime domain G. This hThe full-length calmegin protein contains a signal sequence at its N-terminus for translocation to the endoplasmic reticulum. This signal sequence is missing from the calmegin in the chimera protein, indicating that it is not localized in this cell compartment. Rather, IHC revealed strong intranuclear positivity for BCOR, indicating that not only the wild-type BCOR but the chimera is also localized in the nucleus.Based on our ASA area B and relBCOR-rearranged sarcoma with a novel CLGN fusion that shared morphological and immunophenotypical similarities with other more common fusion variants, which contribute to the continued expanding molecular subtypes of BRS. Molecular ancillary tests, such as NGS and confirmatory Sanger sequencing, serve as powerful tools to discover these unusual variants. In addition, the in silico analysis of the BCOR-CLGN fusion protein is an appropriate approach to aid in better understanding the exact pathogenesis of such a rare variant, via estimation of the fusion protein’s characteristics.In this study, we report the first case of


Stowaway-like MITE, PTE-2, was activated in transgenic Chinese cabbage lines. PTE-2 was revealed by in silico analysis as the putative activated element in transgenic Chinese cabbage lines. To verify the in silico analysis data, MITE insertion polymorphism (MIP) PCR was conducted and PTE-2 was confirmed to be activated in transgenic Chinese cabbage lines. The activation tendency of the copy elements of PTE-2 at different loci was also analyzed and only one more element was activated in the transgenic Chinese cabbage lines. Analyzing the sequence of MIP PCR products, the TSD sequence and TIR motif of PTE-2 were identified and matched to the characteristics of the Stowaway-like MITE family. In addition, the flanking region of PTE-2 was modified when PTE-2 was activated.Transposable elements (TEs) are DNA fragments that can be replicated or transposed within a genome. TEs make up a high proportion of the plant genome and contribute to genetic diversity and evolution, affecting genome structure or gene activity. Miniature inverted-repeat transposable elements (MITEs) are short, non-autonomous class II DNA transposable elements. MITEs have specific sequences, target site duplications (TSDs), and terminal inverted repeats(TIRs), which are characteristics of the classification of MITE families. In this study, a  Transposable elements(TEs) are genetic components that can be replicated or transposed within a genome . TEs canTc1/mariner (DTT), PIF/Harbinger (DTH), hAT (DTA), Mutator (DTM), CACTA (DTC), and P element (DTP) . MIP PCR analysis was used to detect MITEs transposition status , 50 mM EDTA, and 1.25% . MIP PCR was conducted for each locus to identify the activation of copy elements. The primer sets used for the MIP PCR of the copy elements are listed in PTE-2 and its flanking regions were sequenced from the amplicons by MIP PCR. The target amplicon was eluted from the loaded MIP PCR amplicon on a 1% agarose gel using NucleoSpin Gel and a PCR Clean-up Kit . To obtain an accurate consensus sequence for MITEs, the eluted DNA fragment of interest was cloned into the TOPO vector using the MG TA TOPO Cloning kit . The consensus sequence was confirmed by multiple alignments created using CLC Sequence Viewer 8.0 . PTE-2 structure was visualized using the mfold web server , which provides a predictive secondary structure using FASTA format.P-MITE is a database of putative plant MITE information constructed using MITE investigation programs, MITE Digger, MITE-Hunter, and RSPB ,22,23,24PTE-2.Among the MITE mapped on ‘CT001’, SQ041022219 was selected to be activated at the locus of the ‘IGA’ 6 transgenic lines. SQ041022219 from P-MITE was designated PTE-2 loci, which were excised from ‘IGA’ 6 resequencing data, showed an activated status in the MIP PCR test of the T1 ‘IGA’ 6 lines , which provides a predictive secondary structure from the sequence of nucleic acids [PTE-2 secondary structure, which analyzes hairpin-like base pairing with the 5′ and 3′ ends of PTE-2. The TSD sequence was investigated by comparing the excision site of PTE-2-excised transgenic lines with that of the PTE-2-inserted ‘CT001’ lines. The TSD sequence was duplicated and flanked by the PTE-2 sequence at the PTE-2-inserted sequence, whereas the single TSD sequence remained at the PTE-2-excised sequence. The superfamily of the PTE-2 was classified based on its TSD sequence and TIR motif. The CENSOR web server  was used to confirm the similarity to previously described repetitive DNA sequences collected in the Repbase database [To identify the ic acids . The TIRdatabase .PTE-2 had 78% of A+T content and was 268 bp in length. The TSD sequence of PTE-2 was 5’–TA–3’, which is homologous to the Stowaway MITE family 7 microsatellites, were spaced. b’ is a duplicated sequence from the b sequence and it was 9 bp in length. Only eight microsatellites were spaced between the b and b’. c’ is a duplicated sequence from the c sequence and it is 12 bp in length. Between c and c’, 17 bp of sequences, including (T)11 microsatellites, were spaced. d’ is a duplicated sequence from the d sequence, and it was 15 bp in length. Between d and d’, 14 bp of sequences, including (A)8 microsatellites, were spaced.The product of the MIP PCR analysis with the expected A. The amsequence B. The insequence B,C. In FDs) element. The Ds element can move to a new location within the genome only if the activator (Ac) element has provided a transposase (TPase), which is responsible for the transposition event of Ds element [Ac/Ds system represents the relationship between autonomous TEs and non-autonomous TEs. TEs can be divided into two major classes. Class I retrotransposons possess a copy-and-paste transposition mechanism. The class I element is transcribed into mRNA, the intermediate of its transposition, by RNA polymerase II. The mRNA was converted into to cDNA by RT and integrated into the new location of the genome. Because of its transposition event, the retrotransposon element is replicated to a new location, remaining in the donor location. In contrast, class II DNA transposons exhibit a cut-and-paste mechanism. Class II elements are excised from their donor location and moved to a new location by TPase [PTE-2, a class II element, has multiple copies within the genome , and 84.7% of Zea mays [TEs make up a high proportion of the plant genome, accounting for 18.5% of Zea mays . The traZea mays . The TE-Zea mays .B. rapa reference genome were published in the P-MITE database [PTE-2 was classified as the DTT family, the third largest family in B. rapa. Because the Tc1/Mariner superfamily of DNA transposons was considered to be the origin of the Stowaway-like family via its internal deletion [PTE-2 was classified into the Stowaway-like family based on its 2 bp TSD sequence of 5′–TA–3′ and TIR motif homology to the Tc1/Mariner superfamily. Although studies on the activity of MITE belonging to the DTT family are limited, studies have shown that gene expression changes according to the activity of MITE belonging to the DTM family. MnM2, a member of the Mutator family, regulates the MnANR gene associated with the color of tobacco flowers in mulberry trees (Morus notabilis). The expression level of the MnANR gene in transgenic plants was higher than that in the wild type [P-MITE is a database of MITE information for 41 plant species. A total of 174 MITE families, including 1 DTC, 11 DTM, 16 DTT, 56 DTH, and 90 DTA families, in the database . The MITdeletion , PTE-2 wild type .mPing, a MITE in the O. sativa genome, was mobilized in transgenic rice plants [nDaiZ, a member of the hATt family, was activated during tissue culture. In scutellum-induced rice plants, nDaiZ was confirmed to transpose another genomic region through PCR analysis [AhMITE1, was activated during tissue culture. AhMITE1 was activated with a 6.25% transposition frequency in cultivar “Tifrunner” [TEs are activated or repressed under stress conditions . In stree plants . nDaiZ, analysis . In addifrunner” . These rBrassica genome by comparing 20 MITEs that have shown dynamic activity throughout the Brassica genus [PTE-1, which is a MITE in the Brassica genome, was found to be activated during the transformation procedure [PTE-2, PTE-2_c1, and PTE-2_c10 were partially activated in transgenic Chinese cabbage lines  gene increased aluminum toxicity as the gene expression changed [Activated MITEs have been used to analyze the relationship between changes in the characteristic variations in various food crops. In maize, early flowering was induced by MITE insertion in major quantitative trait loci related to flowering time . The MIT changed . As MITE changed  and gent changed .PTE-2 elements were activated, only a single TSD sequence was left at the excised site; however, the sequences flanking the PTE-2-excised site were modified by generating insertions in four regions. It was confirmed that nucleotides A and T remained in the PTE-2 adjacent region when activated .